Modifications of histone tails play a crucial role for the regulation of transcription, telomere maintenance, DNA replication, and chromosome segregation (Kouzarides T, Cell 2007, 128(4): 693-705). Examples of such modifications include acetylation, phosphorylation, methylation, and/or ubiquitination. Covalent modifications in particular regulate not only the chromatin structure but also the interaction with chromatin binding proteins (Kouzarides T, Cell 2007, 128(4): 693-705; Strahl B D and Allis C D, Nature 2000, 403(6765): 41-5; Jenuwein T and Allis C D, Science 2001, 293(5532): 1074-80). In addition, methylation of H3K4, H3K36, and H3K79 is associated with euchromatin structure, whereas that of H3K9, H3K27, and H4K20 is associated with heterochromatin structure (Strahl B D and Allis C D, Nature 2000, 403(6765): 41-5; Jenuwein T and Allis C D, Science 2001, 293(5532): 1074-80).
Conformation of chromatin is one of the key regulators of transcription; untranscribed genes are compacted in heterochromatin, while transcribed genes are in euchromatin, where transcriptional complexes are accessible to the target DNA (Li B et al., Cell 2007, 128(4): 707-19). In addition, modification of histone residue facilitates the interaction with its binding protein(s), and affects subsequent modifications on other histone tails (Strahl B D and Allis C D, Nature 2000, 403(6765): 41-5; Jenuwein T and Allis C D, Science 2001, 293(5532): 1074-80; Li B et al., Cell 2007, 128(4): 707-19; Zhang Y and Reinberg D, Genes & development 2001, 15(18): 2343-60). For example, phosphorylation of H3 serine 10 (H3S10) suppresses methylation of H3K9. Conversely, methylation of H3K9 antagonizes phosphorylation of H3S10. Phosphorylation of H3S10 promotes acetylation of H3K14 by GCN5 (Zhang Y and Reinberg D, Genes & development 2001, 15(18): 2343-60).
The methylation of H3K9 is involved in the transport of HP1 to distinct chromosomal areas, which, in turn, is crucial for establishing and maintaining domains of heterochromatin (Nakayama J et al., Science 2001, 292(5514): 110-3; Lachner M et al., Nature 2001, 410(6824): 116-20; Bannister A J et al., Nature 2001, 410(6824): 120-4). Recruitment of HP1 proteins to certain sites of the genome involves interactions with multiple components of chromatin (Nielsen S J et al., Nature 2001, 412(6846): 561-5). This recruitment is believed to suppress methylation of H3K4, a protein that is crucial for transcriptional activation. These data indicate the complex nature of histone modification that is regulated by the interplay between different modifications. Indeed, a growing number of these proteins have been shown to promote or inhibit tumourigenesis through their histone methyltransferase activity (Varambally S et al., Nature 2002, 419(6907): 624-9; Hamamoto R et al., S Nature cell biology 2004, 6(8): 731-40; Gibbons R J, Human molecular genetics 2005, 14 Spec No 1: R85-92).
Although methylation of histone tails has been intensively studied, that of non-histone protein remains unclear. Recent studies reported that SET7/9, a histone H3K4 MTase, catalyzes TAF10 and p53 as substrates (Kouskouti A et al., Mol Cell 2004, 14(2): 175-82; Chuikov S et al., Nature 2004, 432(7015): 353-60.) Vascular endothelial growth factor receptor-1 (VEGFR1) (Accession No.: NM—002019) is a receptor tyrosine kinase (RTK) that plays a role in physiological and pathological angiogenesis in the context of receptor dimerization and an interaction with its ligands (Shibuya M et al., Oncogene 1990, 5(4): 519-24; Rahimi N, Experimental eye research 2006, 83(5): 1005-16).
VEGFR1 shares structural similarity with the FMS/KIT/PDGFR family, containing an extracellular domain, seven immunoglobulin (Ig)-like sequences, and a cytoplasmic tyrosine kinase domain with a long kinase insert. VEGFR1 is expressed in two forms, as a full-length tyrosine kinase receptor and in a soluble form that carries only the extracellular domain. Through homodimerization or heterodimerization with other RTKs such as VEGFR2 and VEGFR3, the full-length form of VEGFR1 positively mediates signaling on binding with its ligands. However, the soluble form of VEGFR1 acts as an inhibitor through ligand trapping, and suppresses angiogenesis (Shibuya M, Journal of biochemistry and molecular biology 2006, 39(5): 469-78). The regulatory mechanism of these opposite functions remains unclear.
Although one VEGFR ligand, vascular endothelial growth factor-A (VEGFA), associates with both VEGFR1 and VEGFR2, the affinity of VEGFA to VEGFR1 is at least one order of magnitude higher than that to VEGFR2 (Sawano A et al., Cell Growth Differ 1996, 7(2): 213-21). On the other hand, the endogenous tyrosine kinase activity of VEGFR1 is extremely low as compared with that of VEGFR2. Upon binding with VEGFA, these receptors dramatically increase their autophosphorylation levels, and induce growth of endothelial cells (ECs), recruitment of EC progenitors, adhesion of natural killer cells to ECs and monocyte migration (Shibuya M et al., Oncogene 1990, 5(4): 519-24; Rahimi N, Experimental eye research 2006, 83(5): 1005-16; Shibuya M, Journal of biochemistry and molecular biology 2006, 39(5): 469-78). Although several VEGFR1 tyrosine-phosphorylation sites and their potential interacting partners have been described in different expression models (Shibuya M, Journal of biochemistry and molecular biology 2006, 39(5): 469-78), the downstream signaling events remain to be delineated, primarily due to the low biological activity of this receptor.
The present inventors previously reported that SMYD3 (Accession No.: AB057595) has di- and tri-methyltransferase activity on lysine 4 of histone H3 (H3-K4) (See WO 2005/071102, the entire contents of which are incorporated by reference herein). In addition, previous reports demonstrate that elevated SMYD3 expression plays a crucial role in the proliferation of colorectal carcinoma (CRC) and hepatocellular carcinoma (HCC) cells (See WO 2003/027143, the entire contents of which are incorporated by reference herein; see also Hamamoto, R. et al., Nat Cell Biol 6, 731-40 (2004)) and Hamamoto R et al., Cancer Sci 2006, 97(2): 113-8). In particular, over-expression of SMYD3 was shown to result in growth promotion of NIH3T3 cells while the knockdown of endogenous SMYD3 expression in several cancer cells was shown to induce growth inhibition and apoptosis of those cells. The present inventors also have shown that retinoblastoma protein (R1) is methylated through interaction with the SET domain of SMYD3, and that such methylation facilitates phosphorylations of RB1 at threonines 821/826 and serines 807/811 by CDK2/cyclinE or CDK6/cyclinD3 complex in vitro and in vivo (See WO2007/004526, the entire contents of which are incorporated by reference herein).    Patent Citation 1: WO2003/027143 (JP 2005-511023)    Patent Citation 2: WO2004/076623 (JP 2006-519009)    Patent Citation 3: WO2005/071102 (JP 2007-519391)    Patent Citation 4: WO 2006/092958, A1    Patent Citation 5: WO 2007/004526, A2    Non Patent Citation 1: Kunizaki, et al. Cancer Res. 2007 Nov. 15; 67(22):10759-65